Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix: Human Embryonic Stem Cell Protocols

Research output: Chapter in Book/Report/Conference proceedingChapterScientificpeer-review

Abstract

The use of animal products in the derivation and maintenance of human pluripotent stem cells (hPSCs) limits their possible applications in research and in clinics. Thus, one of the major goals in regenerative medicine is the establishment of animal-free conditions to support the culture and differentiation of human stem cells. Human fibroblasts produce an extracellular matrix (ECM) which can be extracted without the use of detergents, sterilized, and then used to coat tissue culture plates.
We have shown that human embryonic stem cells (hESCs) grown on this matrix maintain their pluripotency in the presence of medium conditioned by fibroblast cells, and that these cells maintain expression of surface proteins (SSEA4, Tra1-60, Tra1-81), alkaline phosphatase activity, and specific intracellular markers (Nanog, Oct-4, Tert, FoxD3) in hESCs. This growth system reduces exposure of hPSCs to feeder layers and animal ingredients, thereby limiting the risk of pathogenic contamination and additionally, facilitating their manipulation. Herein we present an improved version of our previous protocol for extracting ECM from human foreskin fibroblast using a different buffer. Our new hypotonic shock method is detergent-free, reduces costs, and preserves the integrity of the extracted ECM. This improved protocol has been validated for undifferentiated-state hPSC maintenance (more than 40 passages), stem cell differentiation, and for cell migration assays.
Original languageEnglish
Title of host publicationHuman Embryonic Stem Cell Protocols
EditorsKursad Turksen
Number of pages22
Volume1307
Place of PublicationNew York
PublisherSPRINGER NEW YORK LLC
Publication date2016
Pages39-60
ISBN (Print)978-1-4939-2667-1
ISBN (Electronic)978-1-4939-2668-8
DOIs
Publication statusPublished - 2016
MoE publication typeA3 Book chapter

Publication series

NameMethods in Molecular Biology
Number1307
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Fields of Science

  • Human embryonic stem cells
  • Human pluripotent stem cells
  • In vitro growth
  • Extracellular matrix
  • Hypo-osmotic lysis buffer
  • 317 Pharmacy
  • 318 Medical biotechnology

Cite this

Sanz-Garcia, A., Stojkovic, M., & Escobedo-Lucea, C. (2016). Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix: Human Embryonic Stem Cell Protocols. In K. Turksen (Ed.), Human Embryonic Stem Cell Protocols (Vol. 1307, pp. 39-60). (Methods in Molecular Biology; No. 1307). New York: SPRINGER NEW YORK LLC. https://doi.org/10.1007/7651_2014_154
Sanz-Garcia, Andres ; Stojkovic, Miodrag ; Escobedo-Lucea, Carmen. / Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix : Human Embryonic Stem Cell Protocols. Human Embryonic Stem Cell Protocols. editor / Kursad Turksen. Vol. 1307 New York : SPRINGER NEW YORK LLC, 2016. pp. 39-60 (Methods in Molecular Biology; 1307).
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Sanz-Garcia, A, Stojkovic, M & Escobedo-Lucea, C 2016, Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix: Human Embryonic Stem Cell Protocols. in K Turksen (ed.), Human Embryonic Stem Cell Protocols. vol. 1307, Methods in Molecular Biology, no. 1307, SPRINGER NEW YORK LLC, New York, pp. 39-60. https://doi.org/10.1007/7651_2014_154

Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix : Human Embryonic Stem Cell Protocols. / Sanz-Garcia, Andres; Stojkovic, Miodrag; Escobedo-Lucea, Carmen.

Human Embryonic Stem Cell Protocols. ed. / Kursad Turksen. Vol. 1307 New York : SPRINGER NEW YORK LLC, 2016. p. 39-60 (Methods in Molecular Biology; No. 1307).

Research output: Chapter in Book/Report/Conference proceedingChapterScientificpeer-review

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AB - The use of animal products in the derivation and maintenance of human pluripotent stem cells (hPSCs) limits their possible applications in research and in clinics. Thus, one of the major goals in regenerative medicine is the establishment of animal-free conditions to support the culture and differentiation of human stem cells. Human fibroblasts produce an extracellular matrix (ECM) which can be extracted without the use of detergents, sterilized, and then used to coat tissue culture plates.We have shown that human embryonic stem cells (hESCs) grown on this matrix maintain their pluripotency in the presence of medium conditioned by fibroblast cells, and that these cells maintain expression of surface proteins (SSEA4, Tra1-60, Tra1-81), alkaline phosphatase activity, and specific intracellular markers (Nanog, Oct-4, Tert, FoxD3) in hESCs. This growth system reduces exposure of hPSCs to feeder layers and animal ingredients, thereby limiting the risk of pathogenic contamination and additionally, facilitating their manipulation. Herein we present an improved version of our previous protocol for extracting ECM from human foreskin fibroblast using a different buffer. Our new hypotonic shock method is detergent-free, reduces costs, and preserves the integrity of the extracted ECM. This improved protocol has been validated for undifferentiated-state hPSC maintenance (more than 40 passages), stem cell differentiation, and for cell migration assays.

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KW - 318 Medical biotechnology

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VL - 1307

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Sanz-Garcia A, Stojkovic M, Escobedo-Lucea C. Growth of Human Pluripotent Stem Cells Using Functional Human Extracellular Matrix: Human Embryonic Stem Cell Protocols. In Turksen K, editor, Human Embryonic Stem Cell Protocols. Vol. 1307. New York: SPRINGER NEW YORK LLC. 2016. p. 39-60. (Methods in Molecular Biology; 1307). https://doi.org/10.1007/7651_2014_154